Systems/Circuits

Functional Imaging of Dentate Granule Cells in the AdultMouse Hippocampus

X Gregor-Alexander Pilz,1* X Stefano Carta,2* Andreas Stäuble,2 Asli Ayaz,2 Sebastian Jessberger,1†

and X Fritjof Helmchen2†

Laboratories of 1Neural Plasticity and 2Neural Circuit Dynamics, Brain Research Institute, University of Zurich, CH-8057 Zurich, Switzerland

The hippocampal dentate gyrus is critically involved in learning and memory. However, methods for imaging the activity of its principalneurons, the dentate gyrus granule cells, are missing. Here we demonstrate chronic two-photon imaging of granule cell populationactivity in awake mice using a cortical window implant that leaves the hippocampal formation intact and does not lead to obviousalteration of animal behavior. Using virus delivery, we targeted expression of genetically encoded calcium indicators specifically todentate gyrus granule cells. Calcium imaging of granule cell activity 600 – 800 �m below the hippocampal surface was facilitated by using1040 nm excitation of the red indicator R-CaMP1.07, but was also achieved using the green indicator GCaMP6s. We found that the rate ofcalcium transients was increased during wakefulness relative to an extremely low rate during anesthesia; however, activity still remainedsparse with, on average, approximately one event per 2–5 min per cell across the granule cell population. Comparing periods of runningon a ladder wheel and periods of resting, we furthermore identified state-dependent differences in the active granule cell population, withsome cells displaying highest activity level during running and others during resting. Typically, cells did not maintain a clear statepreference in their activity pattern across days. Our approach opens new avenues to elucidate granule cell function, plasticity mecha-nisms, and network computation in the adult dentate gyrus.

Key words: awake behaving animals; chronic activity measurements; red-shifted Ca 2� indicator imaging; two-photon imaging

IntroductionThe dentate gyrus (DG), part of the mammalian hippocampus, iscritically involved in essential brain functions underlying certain

aspects of learning and memory (Amaral et al., 2007; Jonas andLisman, 2014). Through in vivo electrophysiological recordingsand optogenetic silencing, the activity of dentate granule cells, themain excitatory neuronal cell population of the DG, has beenassociated with hippocampus-dependent behavioral pattern sep-aration but also pattern completion (Leutgeb et al., 2007; Na-kashiba et al., 2012; Neunuebel and Knierim, 2014; Danielson etal., 2016). These findings indicate a highly diverse contribution ofgranule cells to DG computation that appears to depend on theaddition of newborn granule cells by neurogenic divisions of neu-ral stem/progenitor cells that occurs throughout life in the mam-malian DG (Clelland et al., 2009; Sahay et al., 2011; Spalding et al.,2013).

Whereas substantial progress has been made to study thefunctional properties of individual neurons in hippocampal areaCA1 and deep neocortical layers (Levene et al., 2004; Mizrahi etal., 2004; Ziv et al., 2013; Lee et al., 2014; Rickgauer et al., 2014;Fuhrmann et al., 2015; Sheffield and Dombeck, 2015), activitypatterns in the granule cell population and the contribution of

Received Aug. 14, 2015; revised April 28, 2016; accepted May 24, 2016.Author contributions: F.H., S.J., G.A.P., and S.C. designed research; G.A.P., S.C., and A.S. performed research; A.A.

and F.H. analyzed data; G.A.P., S.C., and A.S. contributed to data analyses; and S.J., F.H., A.A., G.A.P., and S.C. wrotethe paper.

This work was supported by the European Molecular Biology Organization (EMBO) Young InvestigatorProgram (S.J.), the Novartis Foundation (S.J.), the Swiss National Science Foundation (SNSF) ConsolidatorProgram (S.J.), SNSF Grants 31003A-156943 (S.J.) and 310030-127091 (F.H.), and the SNSF Sinergia ProjectGrant CRSII3_147660/1 (F.H.). G.-A.P. was supported by an EMBO long-term fellowship. We thank L. Egolf forexperimental support, Y. Sych for help with behavioral experiments, M. Ohkura and J. Nakai for R-CaMP1.07plasmid, and J. Sobart and B. Weber for Cre-dependent R-CaMP1.07 virus.

*G.-A.P. and S.C. contributed equally to this work.†S.J. and F.H. contributed equally to this work.The authors declare no competing financial interests.Correspondence should be addressed Sebastian Jessberger or Fritjof Helmchen, Brain Research Institute,

University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland. E-mail: jessberger@hifo.uzh.chor helmchen@hifo.uzh.ch.

DOI:10.1523/JNEUROSCI.3065-15.2016Copyright © 2016 the authors 0270-6474/16/367407-08$15.00/0

Significance Statement

We describe a technique that allows for chronic, functional imaging of dentate gyrus granule cells in awake, behaving mice in anintact hippocampal circuitry using genetically encoded calcium indicators. This novel approach enables the analyses of individualgranule cell activity over time and provides a powerful tool to elucidate the mechanisms underlying structural and functionalplasticity of the adult dentate gyrus.

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adult-generated granule cells to DG func-tion remain poorly understood. This ispartly due to the lack of optical imagingmethods for resolving granule cell activitywithin the intact hippocampal circuit invivo, given that the DG is located severalhundred microns deep below the ventralborders of the neocortex, which makesoptical access to the DG difficult withoutinterfering with or damaging overlyinghippocampal structures. Consequently,DG granule cells have hardly been re-solved structurally in vivo and have beenimaged functionally in only one study sofar (Gu et al., 2014; Kawakami et al., 2015;Danielson et al., 2016). Functional imag-ing of granule cell ensembles is, however,a prerequisite to further reveal fundamen-tal principles of DG computation duringencoding and expression of hippocampalengrams.

Here, we demonstrate chronic imagingof DG granule cell activity in the intacthippocampus in anesthetized and awakebehaving mice by combining a chroniccortical window preparation with long-wavelength two-photon excitation of ei-ther the red fluorescent protein calciumindicator R-CaMP1.07 (Ohkura et al.,2012) or the green fluorescent protein cal-cium indicator GCaMP6s (Chen et al.,2013). We confirm the sparseness of gran-ule cell activity and demonstrate thatactivity patterns of DG granule cell popu-lations are heterogeneous, depend on behavioral state, and varyflexibly across days.

Materials and MethodsAnimals and R-CaMP1.07/GCaMP6s expression. All experimental proce-dures were conducted in accordance with the ethical principles andguidelines for animal experiments of the Veterinary Office of Switzerlandand were approved by the Cantonal Veterinary Office in Zurich. We usedtransgenic mice with dense regional expression of Cre recombinase inlayer 5 cortical pyramidal neurons and hippocampal granule cells (Rbp4-KL100 BAC-cre line; Mutant Mouse Resource and Research Center No.031125-UCD; Gerfen et al., 2013). To conditionally express R-CaMP1.07in DG granule cells, we injected AAV1-EF�1-DIO-R-CaMP1.07 viruses(�1 � 10 13 vg/ml) into the DGs of 5- to 8-week-old male and femaleRbp4-KL100 BAC-cre mice (n � 9; Ohkura et al., 2012). In another set offour mice, we injected AAV9-CAG-FLEX-GCaMP6s (Chen et al., 2013)into Rbp4-KL100 BAC-cre mice to express this green calcium indicatorin DG granule cells. Coordinates for stereotactic injections of adeno-associated virus (AAV) particles were as follows (from bregma, in mm):�2.0 AP, �1.5 ML, and �2.3 DV from the skull surface.

Cranial window preparation. Cranial window preparation followed aformer description (Dombeck et al., 2010) and was performed underisoflurane anesthesia with body temperature being maintained at �37°Cusing a regulated heating blanket and a rectal thermal probe. The eyes ofthe mouse were covered by Vitamin A cream (Bausch & Lomb) duringthe surgery. After disinfection with Betadine, the skin was opened with ascalpel, and the exposed cranial bone was cleaned of connective tissueand dried with cotton pads (Sugi). A circular piece of cranial bone (Ø 3mm) was removed using a dental drill with the injection site marking thecenter of the circle. Following the insertion of a biopsy punch (Ø 3 mm;Miltex) 1 mm deep into the cortical tissue for 2 min, the cortical tissue

was aspirated with a cut 27 gauge needle connected to a water jet pump.The cortical tissue was gently aspirated, while constantly being rinsedwith Ringer solution, until the white matter tracts of the corpus callosumbecame visible. A stainless-steel cannula (Ø 3 mm, 1.5 mm height) cov-ered by a cover glass (Ø 3 mm, 0.17 mm thickness) was inserted andsecured in place by UV curable dental acrylic cement (Ivoclar Vivadent).To ensure reproducible positioning of the mouse by head fixation underthe microscope objective, a small aluminum hook was glued to the skullon the contralateral side of the head with dental cement. Five days afterthe surgery, mice were first habituated to the experimenter by handling.Once familiar, animals were trained and accustomed to running on arunning wheel placed under the two-photon microscope while beinghead fixed. The running speed of the mice was recorded in real time witha rotary encoder (Distrelec rotary encoder, incremental 5 VDC, 360,RI32-O/360AR.11KB, Hengstler) in parallel with calcium imaging ofgranule cell population activity.

Immunohistochemistry and confocal microscopy. After the last in vivoimaging session, animals received a lethal dose of pentobarbital (Es-konarcon, Streuli), were flushed with NaCl (0.9%, sterile) until the liverturned pale, and subsequently were transcardially perfused with 4%paraformaldehyde (PFA; 0.1 M phosphate buffer, pH 7.4). Brains werepostfixed in 4% PFA (overnight at 4°C) and dehydrated in 30% sucrose(0.1 M phosphate buffer). Brains were cut into 40 �m free-floating sec-tions using a vibratome (Leica VT1200s). Immunohistochemistry using�-Prox1 primary antibody (rabbit, 1:2000; Millipore Bioscience Re-search Reagents) was performed as described previously (Karalay et al.,2011). A secondary antibody conjugated to Alexa Fluor 488 or Cy3 wasused to visualize Prox1. R-CaMP1.07 and GCaMP6s were not amplifiedby antibody staining, as the signal from these fluorescent proteins re-mained stable and strong after fixation and cutting. Fluorescence imagesof brain sections were acquired with a confocal laser-scanning micro-scope (Olympus FV1000) using 546 and 488 nm laser lines to excite

Figure 1. Imaging of genetically encoded calcium indicators expressed in DG granule cells of mouse hippocampus. A, Schematicillustration of experimental setup showing the chronic window implant above the intact corpus callosum and area CA1 of thehippocampus that allows for red and green calcium indicator imaging in the intact DG/hippocampal circuit. B, Coronal section ofthe fixed brain after in vivo imaging sessions. Note the preservation of hippocampal areas CA1 and CA3 (slight distortion of the brainis due to the removal of the cannula required for the chronic cranial window). C, Selective expression of R-CaMP1.07 (unamplifiedsignal from R-CaMP1.07 protein; magenta) and GCaMP6s (unamplified signal from GCaMP6s protein; green) in DG granule cellswas confirmed by colabeling with nuclear DAPI (gray, top) and the granule-cell-specific transcription factor Prox1 (cyan, bottom).Scale bars: B, 1 mm; C, top, 50 �m; bottom, 20 �m.

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R-CaMP1.07 and GCaMP6s, respectively. Image analysis was performedusing Fiji software (Schindelin et al., 2012).

Behavioral testing. For open-field behavioral testing, unoperatedanimals were placed in the center of a squared arena (45 � 45 � 40cm) made from gray Plexiglas that was illuminated from a centereddiffuse light source. Single animals were allowed to explore the openfield for 10 min while being recorded by a video camera placed abovethe open field and operated by LabView (National Instruments).Movies were analyzed with MATLAB, extracting the distance traveledby the animals and the frequency of rearing events as assessed bymanual scoring. After the first behavioral testing, six animals receivedviral injections (R-CaMP1.07 or GCaMP6s) into the DG and hip-pocampal window implantation, whereas four control animals re-ceived viral injections but no window implant. Two weeks afterimplantation of the hippocampal window, open-field testing was re-peated for both the operated and the sham-operated group. Analysiswas performed using paired or unpaired t test (Excel) for intraindi-vidual and group comparisons, respectively.

Two-photon calcium imaging. We used a custom-built, two-photonmicroscope of the Sutter Instrument Movable Objective Microscopetype, equipped with a 16� long-working-distance, water-immersion ob-jective (0.8 numerical aperture, model CFI75 LWD 16�W, Nikon), aPockels cell (model 350/80 with controller model 302RM, Conoptics),and galvanometric scan mirrors (model 6210, Cambridge Technology),controlled by HelioScan software (Langer et al., 2013). For excitation ofR-CaMP1.07, we used a ytterbium-doped potassium gadolinium tung-state (Yb:KGW) laser (1040 nm, �2 W average power, �230 fs pulses at80 MHz; model Ybix, Time Bandwidth Products). GCaMP6s was excitedat 920 nm with a standard Ti:sapphire laser system (�100 fs laser pulsewidth; Mai Tai HP, Newport). Fluorescence was collected with a redemission filter (610/75 nm; AHF Analysetechnik). In experiments underanesthesia, the average laser power beneath the objective for excitingR-CaMP1.07 and GCaMP6s in the granule cell layer was 239 � 104 mW

and 141 � 44 mW, respectively (mean � SD). In awake experiments, theaverage laser power was 234 � 75 mW and 133 � 50 mW, respectively.We estimate that the laser power in the focal volume (600 – 800 �m deepin the tissue) was �10 – 40 times lower, assuming a scattering length of200 –250 �m at 920 –1040 nm (Kobat et al., 2009). Laser intensity pre-sumably was also attenuated by scattering in the white matter tract of thecorpus callosum and potentially by clipping of the beam at the windowimplant edges.

For imaging experiments under anesthesia, each mouse was anes-thetized with isoflurane (2% in oxygen) and fixed with their alumi-num head post to a holder to keep the animal stable during imaging.Body temperature was constantly monitored and kept at 37°C with aheating pad. Normal Ringer’s solution [containing (in mM) 135 NaCl,5.4 KCl, 5 HEPES, 1.8 CaCl2, pH 7.2] was used as immersion mediumfor the 16� water-immersion objective. For awake experiments, themouse was head fixed under the microscope objective and placed ontop of a running ladder wheel (Ø 20 cm) with regularly spaced rungs(1 cm spacing). Running speed and running distance were recorded at40 Hz with a rotary encoder and synchronized to calcium imaging ofgranule cell populations. In imaging sessions, the activity of calciumindicator-expressing granule cells was recorded in trials of 20 s dura-tion, interleaved with 10-s-long breaks without laser illumination andrecording (maximum number of trials, n � 30). Animals were keptunder these running and recording conditions maximally for 45 minper session.

Data analysis and statistics. Calcium indicator fluorescence signals,F, were analyzed using custom software routines in Fiji, MATLAB(MathWorks), and IGOR (Wavemetrics). We subtracted backgroundfluorescence (estimated as the bottom first percentile fluorescencesignal across the entire movie) and applied a hidden Markov modelline-by-line motion correction algorithm (Dombeck et al., 2007).Trials obviously insufficiently motion corrected were excluded fromthe analysis. In some case, images were spatially smoothed with a

Figure 2. In vivo calcium imaging of DG granule cell activity in anesthetized mice. A, In vivo two-photon images of R-CaMP1.07-expressing DG granule cells in an anesthetized, head-fixed mouse.The right panel shows the imaging field. B, F/F calcium traces for 12 example neurons from the neuronal population shown in A. Example traces are shown for granule cells displaying no activity(gray) or for cells exhibiting large F/F transients (red). Red arrows in A correspond to the two neurons active in this session. C, Percentage of R-CaMP1.07-expressing cells showing activity duringisoflurane anesthesia. D, In vivo two-photon images of GCaMP6s-expressing DG granule cells in an anesthetized, head-fixed mouse. The right panel shows the imaging field. E, F/F calcium tracesfor 13 example neurons from the neuronal population shown in D. Example traces for inactive (gray) and active (red) granule cells. Red arrows in D correspond to the three neurons showing largecalcium transients in this session. F, Percentage of GCaMP6s-expressing granule cells showing activity during isoflurane anesthesia. Scale bars: A, D, left, 50 �m; right, 20 �m. dpi, Dayspostinjection.

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Gaussian filter (1 pixel width). Regions ofinterests corresponding to individual neu-rons were manually selected from the meanimage of a single-trial time series.Background- and motion-corrected calciumsignals were expressed as the relative per-centage change, F/F � (F � F0)/F0, whereF0 was calculated as the eighth percentile ofthe distribution of fluorescence values. F/Ftraces were smoothed with a five-point, first-order Savitsky–Golay filter. “Large” calciumtransients were detected based on the follow-ing criteria: First, a F/F baseline was calcu-lated as the fifth percentile of the meanfluorescence in an 8 s sliding window, andbaseline noise was taken as the first percen-tile of the SD values obtained in an 8 s slidingwindow. Fluorescence transients were con-sidered large when their peak amplitude de-viated from baseline more than three timesthe baseline noise for R-CaMP1.07 (fourtimes for GCaMP6s). Second, the peak F/Famplitude had to be at least 25%. Third, twoconsecutive peaks had to be separated by atleast 800 ms to avoid detection of calciumtransient shoulders.

To quantify the decay kinetics of calciumtransients, we selected isolated salient calciumtransients (�50% F/F ) and fitted an expo-nential curve to the decay phase, revealing asignificantly faster time constant for granulecells expressing R-CaMP1.07 compared tothose expressing GCaMP6s (1.47 � 0.71 s vs2.12 � 1.26 s; n � 38 and 47 events, respec-tively; mean � SD; p 0.01, unpaired t test).As our analysis was focused on detection ofcalcium transients rather than their timecourse, the results are nonetheless consistent for the two indicators.

We counted the frequency of large calcium transients across differentbehavioral conditions. Running speed was downsampled to the imagingframe rate (10 Hz); periods with speeds larger than 0.5 cm/s were con-sidered “running” periods, and periods with speeds between �0.5 and0.5 cm/s were considered “rest” periods. Values below �0.5 cm/s wereignored and not included in either running or resting periods. Cells witha calcium transient frequency larger than 0.01 min �1 were consideredactive and otherwise were considered inactive. Active cells were furtherclassified as being preferentially active in running or resting periods, or inboth, as follows: We resampled our fluorescence data using a modifiedbootstrapping method. Randomly sampled 1-s-long traces were concat-enated to create fluorescence traces of the same length of the originaldata. We repeated this 100 times and computed the mean frequency ofevents during running and resting periods (frun, frest) as well as their SDvalues (�run, �rest). As a sensitivity index, we calculated the d� value:

d� �frun � frest

�12��run2 � �rest2 .

Cells with d� � 1.8 were considered run cells, cells with d� �1.8 wereconsidered rest cells, and cells with d� between �1.8 and 1.8 were labeledas being active during both conditions. Any active cell for which the totalduration of either run or resting periods was 5% of the recording timewas labeled as “uncharacterized” because no reliable statement was pos-sible about preference in this case. Characterization was based on theactivity on individual imaging days. Pie charts of neuronal preference(see Fig. 4A) were computed using data from only the first day of record-ing of each neuron. Repeated measurements from the same neurons werenot pooled. Statistical analysis was performed using an unpaired t test(Excel).

Responses to either locomotion start or end were evaluated by firstaligning neuronal calcium traces to the onset and offset of running peri-ods, respectively, and averaging across all instances for both conditions.If the peak amplitude (after smoothing with a nine-point Savitzky–Golayfilter) in a 3 s time window following the transition exceeded five timesthe SD in a 0.9 s pretransition window, the response was consideredsignificant.

ResultsCell type-specific expression of genetically encoded calciumindicators in dentate granule cellsTo allow for functional imaging of DG granule cells, we expressedeither the red fluorescent calcium indicator R-CaMP1.07 (Oh-kura et al., 2012) or the green fluorescent calcium indicatorGCaMP6s (Chen et al., 2013) in granule cells by injecting a Cre-dependent AAV into the DGs of transgenic mice expressing Crerecombinase under the control of the regulatory elements of theretinol binding protein 4 (Rbp4) gene that is highly expressed indistinct DG granule cells (and other neuronal cells outside theDG such as layer 5 cortical neurons; Gerfen et al., 2013). Thisapproach resulted in specific labeling of DG granule cells withinthe adult hippocampus as verified by post hoc histological stainingusing Prox1, a homeobox-domain transcription factor that isselectively expressed in granule cells in the adult mouse forebrain(Karalay et al., 2011; Fig. 1A–C; see Materials and Methods). Toprovide optical access to the hippocampus for in vivo imaging, welocally removed overlying cortical tissue by aspiration and subse-quently implanted a chronic window (Mizrahi et al., 2004; Dom-beck et al., 2010; Gu et al., 2014). The hippocampus itself andthe overlying corpus callosum remained unharmed to avoid

Figure 3. In vivo calcium imaging of DG granule cell activity in awake mice. A, In vivo two-photon images of R-CaMP1.07-expressing DG granule cells in an awake, head-fixed mouse running on a ladder wheel. B, F/F calcium traces at rest andduring running periods (shaded areas) for nine example neurons from the neuronal population shown in A (cell numbersshown as subscripts in A). Neuronal traces displaying large F/F transients are marked in red, and the run speed is denotedin blue. Note the enhanced activity in the awake state and the similar activity of individual neurons during the restingperiod. Detected large calcium transients are depicted with blue asterisks. C, Percentage of R-CaMP1.07-labeled granulecells in the DG displaying calcium activity during awake behavior. D, In vivo two-photon images of GCaMP6s-expressing DGgranule cells in an awake mouse. E, F/F calcium traces at rest and during running periods for nine example neurons fromthe neuronal population shown in D (cell numbers are indicated in D). Note the number of cells being active during runningperiods. Detected large calcium transients are depicted with blue asterisks. F, Percentage of GCaMP6s-labeled granule cellsdisplaying calcium activity during the awake state. Scale bars: 20 �m.

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interference with hippocampal/entorhinal cortex (EC) circuitintegrity (Bonnevie et al., 2013). We assessed potential behav-ioral effects in an open-field test. Window implantation af-fected neither the distance traveled in 10 min (3.55 � 0.76 mbefore, 3.57 � 1.39 m after, mean � SD; n � 6; p � 0.9, pairedt test) nor the frequency of rearing events (4.45 � 1.27 min �1

before, 4.28 � 2.27 min �1 after; p � 0.8). These values werealso not significantly different from sham-operated mice (n �4; p � 0.1, unpaired t test).

R-CaMP1.07 and GCaMP6s allow for in vivo granulecell imagingTwo-photon excitation at 1040 nm with a fixed-wavelengthfemtosecond laser allowed for imaging of R-CaMP1.07-expressing granule cells at 600 – 800 �m below the corpus cal-losum (Fig. 2A–C). GCaMP6s excited with a tunable Ti:sapphire laser at 920 nm could also be robustly detected in thegranule cell layer of the DG (Fig. 2D–F ). We first measuredcalcium transients in granule cell populations in anesthetizedmice (n � 7). Consistent across mice, only a small fractionof granule cells displayed occasional large F/F transients(�25%; see Material and Methods) over 1–3 min recordingperiods (4% of R-CaMP1.07-positive cells, n � 695 cells from16 imaging areas of 5 mice; 8% of GCaMP6s-positive cells, n �238 cells from 4 imaging areas from 2 mice; Fig. 2 B, E). Theselarge calcium signals presumably reflect bursts of action po-

tentials, given estimates of 5–15% F/F changes for singleaction potential-evoked R-CaMP1.07 transients in neocorticalpyramidal neurons (Ohkura et al., 2012; Inoue et al., 2015; ourunpublished data). Because the sensitivity of R-CaMP1.07 and

Figure 4. Heterogeneous association of dentate granule cell activity in awake mice with locomotion. A, Preferential activity during running and resting periods of R-CaMP1.07-expressing (top pie chart, n�479 cells from 9 animals) and GCaMP6s-expressing DG granule cells (bottom pie chart, n � 363 cells from 4 animals). Activity preference of cells was classified based on a d� selectivity index (see Materials andMethods). Running (blue) and resting (red) cells showed clear preference for one state (absolute d�� 1.8). A fraction of granule cells exhibited similar rates of activity during both running and resting periods(pink). Granule cells labeled “uncharacterized” showed periods of either resting or running that were too short for a preference analysis. B, C, Mean frequency of largeF/F transients in DG granule cells duringanesthesia and during resting or running in awake mice across the entire data sets for R-CaMP1.07 (B) and GCaMP6s (C). D, E, Distribution of peak amplitudes of large F/F calcium transients in DGgranule cells during resting and running periods for R-CaMP1.07 (D) and GCaMP6s (E). *p 0.05; **p 0.01; ***p 0.001. Error bars indicate SE.

Movie 1. Example movie of in vivo DG granule cell activity, corre-sponding to the recording shown in Figure 3A (28 d postinjection). F/Fchanges are overlaid in green. The right yellow bar indicates the mo-mentary running speed. Movie speed, 20� accelerated. Scale bar, 20�m.

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GCaMP6s for reporting action potential-evoked calcium tran-sients has not been calibrated in DG granule cells so far, wecannot exclude that low-frequency baseline spiking remain-ed undetected. Nonetheless, results obtained with bothR-CaMP1.07 and GCaMP6s in anesthetized mice indicate verylow spiking activity of adult granule cells, in agreement withprevious reports that showed relative sparseness of DG activityusing in vivo electrophysiological recordings and during invivo imaging (Jung and McNaughton, 1993; Leutgeb et al.,2007; Alme et al., 2010; Danielson et al., 2016).

R-CaMP1.07 and GCaMP6s allow granule cell imaging inawake miceAfter showing that imaging of dentate circuits is feasible in anes-thetized mice, we next aimed to assess DG granule cell activity inawake mice during various behavioral states. Mice were adaptedto head fixation and to running on top of a ladder wheel (seeMaterials and Methods). We acquired two-photon image seriesof R-CaMP1.07 fluorescence and GCaMP6s fluorescence in DGgranule cell populations (Fig. 3A–F). Compared to the anesthe-tized condition, a higher fraction of neurons displayed large F/Ftransients for both calcium indicators (Fig. 3C,F; 46% ofR-CaMP1.07-expressing cells; n � 479 cells from 10 imagingareas of 7 mice; 58% of GCaMP6s-expressing cells; n � 363 cellsfrom 5 imaging areas of 4 mice; Fig. 3B,E).

Interestingly, we identified subpopulations of DG granulecells that showed robust activity preferentially during either rest-ing or running (Fig. 3B,E, respectively), suggesting locomotion-associated activity patterns of DG granule cells similar to what hasbeen described previously for CA1 pyramidal cells (McNaughtonet al., 1983; Fuhrmann et al., 2015). Of all cells displaying largecalcium transients, slightly more cells were preferentially activeduring running periods compared to resting conditions (Fig. 4A).Approximately 20% of cells displayed activity in both states, run-ning and resting, in experiments with both R-CaMP1.07 andGCaMP6s (Fig. 4A). Although the frequency of occurrence oflarge calcium transients was significantly higher in awake micethan during anesthesia (Fig. 4B,C), the overall rates across theentire population of about one event per cell every 2–5 min stillindicates sparse DG activity. The amplitude distributions of thecalcium transients revealed a trend of large calcium transientsoccurring more frequently during the resting state for labelingwith R-CaMP1.07. This finding may indicate enhanced bursti-ness of DG granule cells during nonrunning periods (Fig. 4D,Movie 1). However, GCaMP6s-labeled dentate granule cells, incontrast, displayed larger amplitudes during running periods(Fig. 4E). We conclude that granule cells show a spectrum ofbehavior-dependent activity patterns, with distinct preferencesof individual cells.

Figure 5. Repeated imaging of granule cell activity across several days. A, Example calcium traces for three granule cells measured in two separate imaging sessions 1 to several days apart. Twocells were consistently active specifically in either the running state (top row) or during resting (middle row); the cell in the bottom row changed its activity pattern from being active in resting statesto being active during both states. B, Summary plot depicting the specificity of activation pattern for all granule cells repeatedly imaged in three consecutive sessions across several days pooled fromR-CaMP1.07 and GCaMP6s experiments (n � 228 cells). Activity preference of cells was classified based on a d� selectivity index as running (blue), resting (red), or both (pink), or remaineduncharacterized (light gray). Inactive cells are depicted in dark gray (see Materials and Methods). Shading was applied according to the absolute d� value to indicate strength of selectivity. C, Exampletraces for three individual, chronically imaged GCaMP6s-expressing cells from one imaging area exhibiting significant calcium responses aligned to the start (Cells 1 and 2, top and middle rows,respectively) or end (Cell 3, bottom row) of running activity. Note that cells either showed a consistent activation at the start (top) or end (bottom) of running periods in multiple sessions across daysor displayed changes in their activation pattern like the example neuron in the middle row. Scale bars, 30 �m.

7412 • J. Neurosci., July 13, 2016 • 36(28):7407–7414 Pilz, Carta et al. • In Vivo Dentate Gyrus Imaging

Chronic imaging of R-CaMP1.07- and GCaMP6s-expressinggranule cells reveals functional flexibility of dentate granulecellsStrikingly, the activity of granule cells could be monitored repeat-edly across several days (Fig. 5A), indicating the required stabilityof in vivo optical imaging that will allow for analyzing the func-tional activation of individual granule cells during distinct expe-riences in awake mice. Using such a chronic imaging approach,we found examples of both consistency in activity in three con-secutive imaging sessions across several days (i.e., neurons spe-cifically active only during resting or running periods) and moreflexible behavior-dependent activity (e.g., neurons changingfrom preferred activity during resting to being active in bothstates; Fig. 5A,B). From Session 1 to Session 2, a fraction of 45%(103 of 228 cells) retained their preference for a behavioral state,whereas from Session 1 to Session 3, this was the case for only25% of cells (58 of 228 cells; Fig. 5B). This finding indicates apronounced flexibility and relatively low stability of behavioral-state-related granule cell activity over prolonged time periods.Interestingly, we also observed neurons that exhibited large cal-cium transients specifically at either the start or end of runningperiods (Fig. 5C; �36% and 10% for locomotion onset and off-set, respectively; n � 209 cells in 3 mice). In several cases, thetemporal locking of their activation to run start or run end wasstable across consecutive imaging sessions, but we also observedcells that changed their activation pattern (Fig. 5C), indicatingfunctional flexibility of dentate granule cells.

DiscussionOur study provides the first proof of principle that the hippocam-pal DG granule cell population is amenable to functional imagingstudies in vivo with an approach that leaves the hippocampalformation intact. This was achieved by using both a red-shiftedcalcium indicator that can be efficiently two-photon excitedabove 1000 nm (Ohkura et al., 2012; Inoue et al., 2015) and also amore conventional green calcium indicator (Chen et al., 2013).Red fluorescent indicators, of which new variants have been in-troduced recently (Dana et al., 2016), are particularly suitable forfacilitating deep tissue imaging. Notably, our chronic windowimplantation allows for repeated imaging in DG in fully awakemice. This will make it possible to study the activity patterns ofgranule cells as well as other targeted neuronal populations in theDG for more complex behaviors, e.g., locomotion or navigationin a virtual space (Dombeck et al., 2010; Harvey et al., 2012). Inaddition, optical imaging of granule cell activity will allow forcharacterization of activation patterns during distinct phases(i.e., encoding vs retrieval) of dentate-dependent learning andmemory tasks adapted to head-fixed experimental settings (Clel-land et al., 2009; Deng et al., 2013). Excitingly, the approachdescribed here suggests that simultaneous imaging in two distinctcolor channels (e.g., combining red and green calcium indica-tors) to test the functional behavior of two select neuronal pop-ulations appears feasible.

Most recently, chronic imaging experiments of newborn andmature granule cells were described that found relatively highrates of remapping both in old and newborn granule cells whenanimals were exposed to different contextual experiences (Dan-ielson et al., 2016). These are exciting data that support the feasi-bility of chronic DG in vivo imaging. However, and in contrast toour approach that leaves the hippocampus intact, Danielson et al.(2016) used a preparation that at least partially lesions hippocam-pal area CA1, which may substantially affect the functionality ofhippocampal/EC circuitries (Bonnevie et al., 2013; Danielson et

al., 2016). Be that as it may, the substantial rate of remapping ofspatial representations (Bonnevie et al., 2013; Danielson et al.,2016) and the flexibility of granule cell activity in relation tobehavioral-state preference (i.e., running vs resting) shown hereboth suggest that granule cell activation patterns in the adultDG change across days, presumably undergoing context- andexperience-dependent plasticity. Together with previously de-scribed approaches allowing for structural imaging of the DG(Gu et al., 2014; Kawakami et al., 2015), the method describedhere will substantially expand the available toolbox to analyzegranule cell activity in an intact hippocampal circuitry to st-udy the mechanisms underlying functional (e.g., experience-induced) and structural (e.g., neurogenesis-associated) plasticityof the adult DG.

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Functional Imaging of Dentate Granule Cells in the Adult Mouse HippocampusIntroductionMaterials and MethodsResultsR-CaMP1.07 and GCaMP6s allow granule cell imaging in awake miceDiscussion